The present invention relates to deletion mutants of the IL-6 receptor protein, particularly of the beta chain (gp130) of the IL-6 receptor protein, to DNA encoding said protein, and to RNA derived therefrom. Moreover, the invention relates to substances which specifically block the binding of gp130 to Hck as well as to pharmaceutical preparations containing said substances in an amount effective to treat multiple myeloma.
All literature citations in this specification are hereby incorporated herein by reference.
IL-6 is an important growth factor for multiple myelomas (MM). For this reason, mechanisms by which IL-6 induces cell growth have been studied. It has previously been shown that IL-6 induces the activation and tyrosine phosphorylation of Hck, a tyrosine kinase of the Src family. It has been demonstrated that in MM cell lines Hck binds to the IL-6 receptor β chain gp130. A series of gp130 deletion mutants has been constructed on the basis of a chimeric receptor comprising the extracellular portion of the erythropoietin receptor and the intracellular portion of gp130. Cloning of gp130 is described by Hibi, M., et al., “Molecular cloning and expression of an IL-6-signal transducer, gp130,” CELL, 63:1149–1157 (Dec. 21, 1990). Surprisingly, the deletion of a region of 41 amino acids in length between residues 771 and 811 of gp130 (Δ771-811) results in complete disruption of Hck binding to gp130. A striking feature of this region is its remarkably high content of negatively charged residues; for this reason it has been also referred to as an “acidic domain”. Since the Δ771-811 deletion is localized between the two STAT3 binding sites at residues tyrosine (Y) 814 and 767, point mutations have been generated at these sites; this was done to exclude any effect of Y814 or Y767 on the binding of gp130 to Hck. STAT3 activation by gp130 was not affected by the Δ771-811 mutant. Eventually, the stable transfection of receptor mutants into a growth factor-dependent pro B cell line, Baf-B03, showed that deletion Δ771-811 significantly reduced the proliferative response of cells to gp130 stimulation. In conclusion, it has been shown for the first time that Hck binds to an acidic domain of gp130 which is critical for mediating the proliferative response via growth factor-activated gp130.
Interleukin-6 is a major growth factor for MM cells in vitro and in vivo Klein, B., et al., “Interleukin-6 in human multiple myeloma,” Blood, 85:863 (1995); Hallek, M., et al., “Multiple myeloma: increasing evidence for a multistep transformation process, ” Blood, 91:3 (1998); Klein, B., et al., “Cytokine network in human multiple myeloma,” Hematol. Oncol. Clin. North Am., 6:273 (1992); Klein, B., “Cytokine, cytokine receptors, transduction signals, and oncogenes in human multiple myeloma,” Sem. Hem., 32:4 (1995); Kawano, M., et al., “Autodrine generation and requirement of BSF-2/IL-6 for human multiple myelomas, Nature, 332:83 (1988); Zhang, X. G., et al., “Interleukin-6 is a potent myeloma-cell growth factor in patients with aggressive multiple myeloma,” Blood, 74:11 (1989). Despite evidence for a role of IL-6 in the pathogenesis of MM little is known about the signaling mechanisms responsible for IL-6-mediated cell growth in MM.
To exert these biological effects IL-6 must bind to the IL-6 receptor (IL-6R) composed of two α-chains (IL-6Rα, 80 kDa) and two β chains (IL-6Rβ or gp130, 130 kDa). Two moieties of IL-6 and two pairs of these receptor chains form a functional hexameric IL-6R complex. Simpson, R. J., et al., “Interleukin-6: structure-function relationships,” Protein Sci, 6:929 (1997); Somers, W., et al., “A crystal structure of interleukin 6: implications for a novel mode of receptor dimerization and signaling,” Embo J., 16:989 (1997); Ward, L. D., et al., “Influence of interleukin-6 (IL-6) dimerization on formation of the high affinity hexameric IL-6:receptor complex,” J. Biol. Chem., 271:20138 (1996). The subsequent intracellular signaling events are activated by gp130. Activation of IL-6R stimulates at least two major signal transduction pathways, the Ras/mitogen activated protein kinase (MAPK) signaling cascade (Neumann, C., et al., “Interleukin-6 induces tyrosine phosphorylation of the Ras activating protein Shc and its complex formation with Grb2 in the human multiple myeloma cell line LP-1,” Eur. J. Immunol., 26:379 (1996); Ogata, A. et al., “IL-6 triggers cell growth via the Ras-dependent mitogen-activated protein kinase cascade,” J. Immunol, 159:2212 (1997); Takahashi-Tezuka, M., et al., “Gab1 acts as an adapter molecule linking the cytokine receptor gp130 to ERK mitogen-activated protein kinase,” Mol. Cell. Biol, 18:4109 (1998); Shi, Z. Q., et al., “The Shp-2 tyrosine phosphatase has opposite effects in mediating the activation of extracellular signal-regulated and c-Jun NH2-terminal mitogen-activated protein kinases,” J. Biol. Chem., 273:4904 (1998); and Boulton, T. G., et al., “Ciliary neurotrophic factor/leukemia inhibitory factor/interleukin 6/oncostatin M family of cytokines induces tyrosine phosphorylation of a common set of proteins overlapping those induced by other cytokines and growth factors,” J. Biol. Chem., 269:11648 (1994)) and the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway (Berger, L. C., et al., “Tyrosine phosphorylation of JAK-TYK kinases in malignant plasma cell lines growth-stimulated by interleukins 6 and 11,” Biochem. Biophys. Res. Commun., 202:596 (1994); Ihle, J. N., et al., “Signaling by the cytokine receptor superfamily: JAKs and STATs,” Trends. Biochem. Sci., 19:222 (1994); Stahl, N., et al., “Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6β receptor components,” Science, 263 (1994); Gerhartz, C., et al., “Differential activation of acute phase response factor/STAT3 and STAT1 via the cytoplasmic domain of the interleukin-6 signal transducer gp130,” J. Biol. Chem., 271:12991 (1996)). However, the signaling cascades mediating IL-6 induced cell growth are not well defined. It was shown earlier that JAK and STAT proteins are activated by IL-6 in MM cells independently of the proliferative response. In contrast, MAPK was only activated in cells and cell lines showing a proliferative response to IL-6 (Ogata, A. et al., J. Immunol., 159:2212 (1997), cited above).
The inventors herein have previously shown that at least three members of the Src-family of tyrosine kinases, Fyn, Hck, and Lyn, bind to gp130 in MM cells. Hallek, M., et al., “Signal transduction of interleukin-6 invlves tyrosine phosphorylation of multiple cytosolic proteins and activation of Src-family kinases Fyn, Hck, and Lyn in multiple myeoloma cell lines,” Exp. Hematol., 25:1367 (1997). Stimulation of cells with IL-6 increased the activity of these kinases.
It is an object of the present invention to provide means for the inhibition or at least significant reduction of the stimulation of multiple myeloma cells by IL-6 in order to at least significantly decrease the proliferation of tumor cells and particularly of mycloma cells.
The present invention provides an IL-6 receptor protein having a deletion in the region of the beta chain which at least comprises amino acids 771-811. In further studies, this region may be restricted further to reveal smaller deletion regions within the 771-811 region which inhibit the binding of Hck. Preferably, the binding of Hck to gp130 is reduced by more than 90%. It is to be understood however that a larger region of gp130 which comprises the amino acid sequence mentioned above or portions thereof may also be deleted. Preferred deletion mutants are those that comprise the region of amino acids 771-811 or portions thereof.
The present invention resides not only the IL-6 receptor protein having the above-mentioned deletion in gp130 but also in gp130 itself carrying the deletion in the region of 771-811 or portions thereof. The present invention also resides in the IL-6 receptor protein and gp130 with deletions extending beyond the 771-811 region and yet still inhibiting the interaction between Hck and gp130. The invention further resides in DNA encoding the IL-6 receptor proteins characterized above or the mutant gp130 as well as RNA derived from such DNA.